Cerberus/coco derivatives and uses thereof

ABSTRACT

The invention relates to Cerberus/Dan/Gremlin polypeptides or variants thereof for use in treating a variety of disorders associated with myostatin, nodal and GDF-11. Preferred polypeptides are Coco or Cerberus derivatives.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/774,585, filed Feb. 22, 2013 (now U.S. Pat. No. 9,045,553), which isa continuation-in-part of U.S. application Ser. No. 12/940,740, filedNov. 5, 2010 (now U.S. Pat. No. 8,796,199), which is a divisional ofU.S. application Ser. No. 12/001,494, filed Dec. 10, 2007, now U.S. Pat.No. 7,833,971, which claims the benefit of U.S. Provisional Application60/873,933, filed Dec. 8, 2006. U.S. application Ser. No. 13/774,585,filed Feb. 22, 2013, is a continuation-in-part of U.S. application Ser.No. 13/533,733, filed Jun. 26, 2012 (now abandoned), which is acontinuation of U.S. application Ser. No. 11/597,182, filed Mar. 22,2010, (now abandoned), which is a national-stage filing under 35 U.S.C.371 of International Application PCT/US05/18928, filed May 27, 2005,which claims the benefit of U.S. Provisional Application Ser. No.60/575,062, filed May 27, 2004. The specifications of each of theforegoing applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 1, 2015, isnamed PHPH-P06-005_SEQ.txt and is 46,787 bytes in size.

BACKGROUND

Transforming growth factor-β superfamily proteins represent a largefamily of cytokines that includes the TGF-βs, activins, inhibins, bonemorphogenetic proteins (BMPs) and Mullerian-inhibiting substance (MIS)(for review, see Massague et al., Trends Cell Biol. 7:187-192, 1997).These proteins contain a conserved C-terminal cystine-knot motif, andserve as ligands for diverse families of plasma membrane receptors.Members of the TGF-β family exert a wide range of biological effects ona large variety of cell types. For example, they regulate cell growth,differentiation, matrix production and apoptosis. Many of them haveimportant functions during embryonal development in pattern formationand tissue specification; in the adult, they are involved in processessuch as tissue repair and modulation of the immune system.

Activities of the TGF-β superfamily proteins are regulated throughvarious means. One of the negative regulations for the BMP subfamily ofproteins is through a relatively large family of so-called BoneMorphogenetic Protein (BMP) antagonists/repressors. These BMP repressorsrepresent a subgroup of proteins that bind BMPs, and interfere with BMPbinding to their membrane receptors, thereby antagonizing their actionsduring development and morphogenesis.

The BMP repressors can be further divided into three groups of proteinsbased on analysis, especially the number of structurally conserved Cysresidues in their C-terminal characteristic “Cys-knot” structures: the8-, 9-, or 10-member ring Cys-knot BMP repressors. The 8-member ring(CAN subfamily) repressors can be divided further into four subgroupsbased on a conserved arrangement of additional cysteine residues—gremlinand PRDC, Cerberus and coco, and DAN, together with USAG-1 andsclerostin. Orthologs of these human BMP antagonists in the genomes ofseveral model organisms have also been identified, and theirphylogenetic relationship has been analyzed (Avsian-Kretchmer and Hsueh,Mol Endocrinol. 18(1): 1-12, 2004. Epub 2003 Oct. 2, incorporated hereinby reference).

Myostatin, or growth/differentiation factor 8 (GDF-8), also belongs tothe transforming growth factor-β (TGF-β) superfamily (McPherron et al.,Nature 387:83-90 (1997)). The human myostatin gene has been cloned(Nestor et al. Proc. Natl. Acad. Sci. 95:14938-43 (1998)), and it hasbeen reported that myostatin immunoreactivity is detectable in humanskeletal muscle in both type 1 and type 2 fibers. With respect tofunction, myostatin may play a role in negatively regulating the growthand development of skeletal muscle (Nestor et al., supra).

A study with myostatin knock-out mice provided the first evidence thatmyostatin is a key negative regulator of muscle development (McPherronet al., Nature 387:83-90 (1997)). In the myostatin null mice, theanimals were significantly larger than wild-type mice and had a largeand widespread increase in skeletal muscle mass. Furthermore, two breedsof cattle, characterized by increased muscle mass, have mutations in themyostatin coding sequence (McPherron et al., Proc. Natl. Acad. Sci.94:12457-61 (1997)). A naturally occurring myostatin reduced-functionmutation in a human child is associated with gross muscle hypertrophyand a family history of exceptional strength. (Williams M S, N Engl JMed. 2004 Sep. 2; 351(10):1030-1; author reply 1030-1.)

Additionally, it should be noted that the serum and intramuscularconcentrations of immunoreactive myostatin are increased in HIV-infectedmen with muscle wasting compared with healthy men, and correlateinversely with the fat-free mass index. These data support thehypothesis that myostatin is a negative regulator of skeletal musclegrowth in adult men and contributes to muscle wasting in HIV-infectedmen (Nestor et al., supra).

In view of the above findings, a need exists for a manner of regulatingmyostatin activity, particularly in individuals who experience musclewasting as a result of a condition or disease state such as, forexample, aging, Autoimmune Deficiency Syndrome (AIDS), MultipleSclerosis, and cancer. The present invention provides methods andcompositions which may be utilized to help individuals with such musclewasting conditions and provides further insight into the regulation ofmyostatin gene expression.

SUMMARY OF THE INVENTION

One aspect of the invention provides a pharmaceutical preparation ofCerberus, Coco or other polypeptide from the Cerberus/Dan/Gremlinsuperfamily (collectively herein “CDG proteins”) for inhibiting thefunction/signaling of Nodal, myostatin and BMP. Exemplary preparationsof the subject CDG polypeptides may include variant Cerberus or Cocosequences that substantially retain the binding affinity of the parentprotein to Nodal, myostatin and/or another BMP (such as BMP-4). Forinstance, the present invention provides pharmaceutical preparations forinhibiting myostatin, comprising a myostatin antagonist protein thatincludes (at least) a myostatin binding domain of a Cerberus/Dan/Gremlinpolypeptide or variant thereof. The myostatin antagonist protein bindsto and neutralizes one or more of nodal and/or myostatin. Preferably,the pharmaceutical preparation is substantially free of pyrogenicmaterials so as to be suitable for administration as a human orveterinarian therapeutic.

These pharmaceutical preparations can be used to reduce the severity ofa pathologic condition, which is characterized, at least in part, by anabnormal amount, development or metabolic activity of muscle or adiposetissue in a subject. For instance, the pharmaceutical preparations ofthe present invention can be administered in an amount effective toprevent, ameliorate or reduce the severity of a wasting disorder, suchas cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wastingsyndrome, muscular dystrophies, neuromuscular diseases, motor neurondiseases, diseases of the neuromuscular junction, and inflammatorymyopathies. These pharmaceutical preparations can also be used to reducethe severity of a pathologic condition, which is characterized, at leastin part, by an abnormal amount, development or metabolic activity ofheterotopic ossification in tissues such as of muscles, tendons, andligaments; Osteoarthritis (OA), including the development of osteophytesand synovial thickening; ovarian cancer; Fibrodysplasia ossificansprogressiva (FOP); Atherosclerosis, especially inflammatory response inearly steps of atherogenesis in lesion-prone areas; andcraniosynostoses.

Another aspect of the invention provides a pharmaceutical preparation ofCDG protein derivative for specifically inhibiting Nodal and/ormyostatin function without substantially compromising BMP (such asBMP-4) signaling (e.g., does not substantially bind BMP-4 or otherBMPs). Exemplary preparations of this aspect of the invention includepolypeptides including the N-terminal truncated versions of Cerberus orCoco, or other fragments that include the cysteine-core. These so-called“N-terminally truncated CDG derivatives” can be used to reduce theseverity of a pathologic condition, which is characterized, at least inpart, by an abnormal amount, development or metabolic activity of muscleor adipose tissue in a subject. For instance, the pharmaceuticalpreparations of the present invention can be administered in an amounteffective to prevent, ameliorate or reduce the severity of a wastingdisorder, such as cachexia, anorexia, DMD syndrome, BMD syndrome, AIDSwasting syndrome, muscular dystrophies, neuromuscular diseases, motorneuron diseases, diseases of the neuromuscular junction, andinflammatory myopathies.

In certain embodiments, the mysotatin inhibitor is a polypeptide thatincludes a myostatin binding domain of a CDG protein. For instance, theCerberus protein variant can be derived from a human, mouse, or otherspecies of Cerberus, including a human or mouse Cerberus variantsequence sharing at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% ormore sequence similarity or identity with the human or mouse Cerberusprotein, and substantially retain the binding affinity of wild-typeCerberus for myostatin. Likewise, the Coco protein variant can bederived from a human, mouse, or other species of Coco, including a humanor mouse Coco variant sequence sharing at least about 50%, 60%, 70%,80%, 90%, 95%, or 99% or more sequence similarity or identity with thehuman or mouse Coco protein, and substantially retain the bindingaffinity of wild-type Coco for myostatin.

In certain related embodiments, the mysotatin inhibitor is a polypeptidethat includes a myostatin binding domain of a CDG protein, whichpolypeptide does not substantially bind BMP-4 (and other BMPs). Forinstance, the myostatin binding domain can be derived from a human,mouse, or other species of N-terminally truncated Cerberus, including ahuman or mouse Cerberus derivative, with amino acid residues startingfrom any one of residues 106-119 of SEQ ID NO. 2 or 8 of US 2002/0164682A1 (see below), and ending at any residue after residue 241 of SEQ IDNo. 2 or 8 of US 2002/0164682 A1, preferably ending at any residuebetween residues 241 and 267 of SEQ ID NO. 2 or 8 of US 2002/0164682 A1(all residue numbers inclusive).

For example, residues 106-119 of human Cerberus (SEQ ID NO:5) is:

(SEQ ID NO: 1) PPGTQSLIQPIDGM 

Residues 241-267 of human Cerberus is:

(SEQ ID NO: 2) CKVKTEHEDGHILHAGSQDSFIPGVSA 

Also included are Cerberus derived variant sequence, e.g., N-terminallytruncated myostatin binding domain of Cerberus that retains myostatinbinding activity but loses other BMP binding activity. Variant sequencesmay be desirable as a way to alter selectivity of the inhibitor (e.g.,relative to GDF-8, GDF-11 or nodal binding), alter other bindingcharacteristics with respect to myostatin (such as K_(d), and/or K_(on)or K_(off) rates), or improve biodistribution or half life in vivo or onthe shelf.

In certain preferred embodiments, the Cerberus polypeptide (full-lengthor N-terminally truncated) comprising the myostatin binding domain bindsmyostatin with a K_(d) of 1 μM or less, and more preferably a K_(d) of100 nM, 10 nM or even 1 nM or less.

In certain related embodiments, the mysotatin inhibitor is a polypeptidethat includes a myostatin binding domain of a Coco protein, such as thehuman Coco protein shown in FIG. 3 or in GenBank Accession number22749329. An exemplary human Coco protein sequence is

(SEQ ID NO: 3)  1 MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSW 61 KAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL121 RNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI181 EGCHCSPKA 

In certain preferred embodiments, the Coco polypeptide (full-length orN-terminally truncated) comprising the myostatin binding domain bindsmyostatin with a K_(d) of 1 μM or less, and more preferably a K_(d) of100 nM, 10 nM or even 1 nM or less.

In certain embodiments, the myostatin binding domain is part of a fusionprotein including, in addition to the myostatin binding domain, one ormore polypeptide portions that enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. Forinstance, the fusion protein can include an immunoglobulin Fc domain.The fusion protein may include a purification subsequence, such as anepitope tag, a FLAG tag, a polyhistidine sequence, or as a GST fusion.

In certain embodiments, the myostatin binding domain is part of aprotein that includes one or more modified amino acid residues, such asa glycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, or an amino acid conjugated to an organicderivatizing agent.

In certain embodiments, the subject variant CDG polypeptide is selectivefor binding and inhibition of myostatin, e.g., relative to GDF-11 and/ornodal. For instance, the variant CDG polypeptide can be one which has adissociation constant (K_(d)) for myostatin binding that is at least 2times less than its K_(d) for binding GDF-11 and/or nodal, and even morepreferably at least 5, 10, 100 or even 1000 times less. Whether byvirture of binding kinetics or biodistribution, the subject variant CDGpolypeptide can also be selected based on relative in vivo potency, suchas an inhibitor that has an EC₅₀ for inhibiting myostatin activity, or aparticular physiological consequence (such as promoting muscle growth,promoting bone density or inducing adipocytes differentiation) that isat least 2 times less than its EC₅₀ for inhibiting GDF-11 and/or nodalactivities, and even more preferably at least 5, 10, 100 or even 1000times less.

In certain embodiments, the subject variant CDG polypeptide is selectivefor binding and inhibition of myostatin, e.g., relative to other BMPproteins such as BMP-4. For instance, the variant CDG polypeptide can beone which has a dissociation constant (K_(d)) for myostatin binding thatis at least 2 times less than its K_(d) for binding BMP-4, and even morepreferably at least 5, 10, 100 or even 1000 times less. Whether byvirture of binding kinetics or biodistribution, the subject variant CDGpolypeptide can also be selected based on relative in vivo potency, suchas an inhibitor that has an EC₅₀ for inhibiting myostatin activity, or aparticular physiological consequence (such as promoting muscle growth,promoting bone density or inducing adipocytes differentiation) that isat least 2 times less than its EC₅₀ for inhibiting BMP-4 activities, andeven more preferably at least 5, 10, 100 or even 1000 times less.

In certain preferred embodiments, the variant CDG polypeptide bindingdomain binds myostatin with a K_(d) of 1 μM or less, and more preferablya K_(d) of 100 nM, 10 nM or even 1 nM or less.

In general, the subject myostatin inhibtor preparations are suitable foruse in a human patients. In preferred embodiments, the subjectpreparations of variant CDG polypeptides will be substantially free ofpyrogenic materials so as to be suitable for administration to a humanpatient.

In other embodiments, the subject variant CDG polypeptides can beadministered to non-human animals, particularly other mammals. Forexample, the compounds of the present invention can be given tochickens, turkeys, livestock animals (such as sheep, pigs, horses,cattle, etc.), companion animals (e.g., cats and dogs) or may haveutility in aquaculture to accelerate growth and improve the protein/fatratio. To further illustrate, the subject variant Cerberus polypeptidescan be used to stimulate growth or enhance feed efficiency of animalsraised for meat production to improve carcass quality, or to increasemilk production in dairy cattle.

Another aspect of the invention relates to packaged pharmaceuticalscomprising a pharmaceutical preparation of a variant CDG polypeptide, asdescribed herein, and a label or instructions for use in promotinggrowth of muscle tissue in a human patient.

Still another aspect of the invention relates to packagedpharmaceuticals comprising a pharmaceutical preparation of a variant CDGpolypeptide, as described herein, and a label or instructions forveternerian use in promoting growth of muscle tissue in a non-humanmammal.

Another aspect of the invention relates to a method for inhibitingmyostatin signal transduction in vivo by administering a pharmaceuticalpreparation of one or more of the subject variant CDG polypeptides. Thesubject method can be used to promote muscle growth, promote adipogenicdifferentiation, and/or promote bone growth or mineralization in humanpatients or in non-human animals.

In certain embodiments, the treatment methods of the present inventioncan be used to reduce the severity of a pathologic condition, which ischaracterized, at least in part, by an abnormal amount, development ormetabolic activity of muscle or adipose tissue in a subject. Forinstance, the pharmaceutical preparations of the present invention canbe administered in an amount effective to prevent, ameliorate or reducethe severity of a wasting disorder, such as cachexia, anorexia, DMDsyndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophies,neuromuscular diseases, motor neuron diseases, diseases of theneuromuscular junction, and inflammatory myopathies.

Exemplary muscular dystrophies that can be treated with a regimenincluding the subject myostatin include: Duchenne Muscular Dystrophy(DMD), Becker Muscular Dystrophy (BMD), Emery-Dreifuss MuscularDystrophy (EDMD), Limb-Girdle Muscular Dystrophy (LGMD),Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (Also known asLandouzy-Dejerine), Myotonic Dystrophy (MMD) (Also known as Steinert'sDisease), Oculopharyngeal Muscular Dystrophy (OPMD), Distal MuscularDystrophy (DD), and Congenital Muscular Dystrophy (CMD).

Exemplary motor neuron diseases that can be treated with a regimenincluding the subject myostatin include: Amyotrophic Lateral Sclerosis(ALS) (Also known as Lou Gehrig's Disease), Infantile Progressive SpinalMuscular Atrophy (SMA, SMA1 or WH) (Also known as SMA Type 1,Werdnig-Hoffman), Intermediate Spinal Muscular Atrophy (SMA or SMA2)(Also known as SMA Type 2), Juvenile Spinal Muscular Atrophy (SMA, SMA3or KW) (Also known as SMA Type 3, Kugelberg-Welander), Spinal BulbarMuscular Atrophy (SBMA) (Also known as Kennedy's Disease and X-LinkedSBMA), and Adult Spinal Muscular Atrophy (SMA).

Exemplary inflammatory myopathies that can be treated with a regimenincluding the subject myostatin include: Dermatomyositis (PM/DM),Polymyositis (PM/DM), and Inclusion Body Myositis (IBM).

Exemplary diseases of the neuromuscular junction that can be treatedwith a regimen including the subject myostatin include: MyastheniaGravis (MG), Lambert-Eaton Syndrome (LES), and Congenital MyasthenicSyndrome (CMS).

Exemplary myopathies due to endocrine abnormalities that can be treatedwith a regimen including the subject myostatin include: HyperthyroidMyopathy (HYPTM) and Hypothyroid Myopathy (HYPOTM).

Exemplary diseases of peripheral nerve that can be treated with aregimen including the subject myostatin include: Charcot-Marie-ToothDisease (CMT), Dejerine-Sottas Disease (DS), and Friedreich's Ataxia(FA).

Other exemplary myopathies that can be treated with a regimen includingthe subject myostatin include: Myotonia Congenita (MC), ParamyotoniaCongenita (PC), Central Core Disease (CCD), Nemaline Myopathy (NM),Myotubular Myopathy (MTM or MM), and Periodic Paralysis (PP).

Exemplary metabolic diseases of muscle that can be treated with aregimen including the subject myostatin include: PhosphorylaseDeficiency (MPD or PYGM), Acid Maltase Deficiency (AMD),Phosphofructokinase Deficiency (PFKM), Debrancher Enzyme Deficiency(DBD), Mitochondrial Myopathy (MITO), Carnitine Deficiency (CD),Carnitine Palmityl Transferase Deficiency (CPT), Phosphoglycerate KinaseDeficiency (PGK), Phosphoglycerate Mutase Deficiency (PGAM or PGAMM),Lactate Dehydrogenase Deficiency (LDHA), and Myoadenylate DeaminaseDeficiency (MAD).

The subject method can also be used to prevent, ameliorate or reduce theseverity of a metabolic disorder, such as in the treatment of obesity ortype II diabetes. To further illustrate, the subject variant CDGpolypeptide preparations can be used to decrease body fat proportion ina subject.

In still other embodiments, the variant CDG polypeptide preparations canbe used as part of such methods as: treating or preventing congestiveheart failure; for reducing frailty associated with aging; increasingbone density (such as for treating osteoporosis) or accelerating bonefracture repair; treating growth retardation, treatment of physiologicalshort stature, attenuating protein catabolic response such as after amajor operation; reducing protein loss due to chronic illness;accelerating wound healing; accelerating the recovery of burn patientsor patients having undergone major surgery; maintenance of skinthickness; metabolic homeostasis and renal homeostasis. Still other usesof the subject variant CDG polypeptides include: treating growth hormonedeficient adults and preventing catabolic side effects ofglucocorticoids.

The subject pharmaceutical composition can also be used as myostatinantagonist to treat a number of neuronal system disease conditions,including CNS injuries/disease such as spinal cord injury and stroke,and PNS injuries/diseases.

The present invention also contemplates the use of the subject myostatinformulations conjointly with one or more other compounds useful in aneffort to treat the diseases or therapeutic indications enumeratedabove. In these combinations, the therapeutic agents and the variant CDGpolypeptides of this invention may be independently and sequentiallyadministered or co-administered. Combined therapy to inhibit boneresorption, prevent osteoporosis, reduce skeletal fracture, enhance thehealing of bone fractures, stimulate bone formation and increase bonemineral density can be effectuated by combinations of bisphosphonatesand the variant CDG polypeptides of this invention. Bisphosphonates withthese utilities include but are not limited to alendronate, tiludronate,dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate,and BM-210995 (ibandronate).

The subject mysostatin inhibitors may be combined with a mammalianestrogen agonist/antagonist. The term estrogen agonist/antagonist refersto compounds which bind with the estrogen receptor, inhibit boneturnover and prevent bone loss. In particular, estrogen agonists areherein defined as chemical compounds capable of binding to the estrogenreceptor sites in mammalian tissue, and mimicking the actions ofestrogen in one or more tissue. Estrogen antagonists are herein definedas chemical compounds capable of binding to the estrogen receptor sitesin mammalian tissue, and blocking the actions of estrogen in one or moretissues. A variety of these compounds are described and referencedbelow, however, other estrogen agonists/antagonists will be known tothose skilled in the art. Exemplary estrogen agonist/antagonists includedroloxifene and associated compounds (see U.S. Pat. No. 5,047,431),tamoxifen and associated compounds (see U.S. Pat. No. 4,536,516),4-hydroxy tamoxifen (see U.S. Pat. No. 4,623,660), raloxifene andassociated compounds (see 4U.S. Pat. No. 4,418,068), and idoxifene andassociated compounds (see U.S. Pat. No. 4,839,155).

The subject mysostatin inhibitors may also be combined with one or moreof the following agents: glutamate antagonists (including partialantagonists) such as riluzole and topiramate; polypeptide growthfactors, such as growth hormone (GH) and insulin-like growth factor 1(IGF-1), or drugs that increases the body's own production ofneurotrophic factors, such as xaliproden; anti-inflammatory agents, suchas celecoxib (Celebrex) and other COX-2 inhibitors; antibiotics, such asminocycline (Minocin, Dynacin) or other agents that inhibit caspaseenzymes; Protein kinase C inhibitors such as tamoxifen (Nolvadex); andvarious over-the-counter substances, including vitamin E, coenzyme Q10and creatine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a full-length and an N-terminallytruncated version of Cerberus, showing the intact C-terminal Cys-knotdomain. The lower panel shows the predicted position of the N-terminaldeletion of Cerberus, based on the crystal structure of Noggin bound toBMP-7.

FIG. 2 shows a schematic drawing of where Wnt, Nodal and BMP bind toCerberus. BMP-2 and the highly related BMP-4 competitively bindCerberus, likely in the same region. Other more distantly related orunrelated proteins, such as TGF-beta1, EGF, and PDGF, do not competewith BMP-4. The N-terminally truncated version of Cerberus still bindsXnr-1 (Xenopus homolog of mouse Nodal).

FIGS. 3A and 3B

Coco, a BMP inhibitor. (A) Nucleotide sequence of Xenopus Coco (SEQ IDNO: 11) and the corresponding translated amino acid sequence (SEQ ID NO:12). (B) Alignment at the amino acid level of Xenopus (SEQ ID NO: 13),Fugu (SEQ ID NO: 16), human (SEQ ID NO: 15) and mouse (SEQ ID NO: 14)Coco and other family members, Xenopus Cerberus (SEQ ID NO: 17), humanCerberus (SEQ ID NO: 18), chicken Cerberus (SEQ ID NO: 19) and chickenCaronte (SEQ ID NO: 20). See Bell et al. (2003) Development 130(7):1381-1389 which is incorporated by reference herein.

FIG. 4 Binding of Caronte to GDF-11. The tracing shows that Carontebinds to GDF-11 on a BiaCore chip. GDF-11 was immobilized on a BiaCoreCM5 chip using standard amine coupling procedure. Trace: Caronte (200μg/ml; R&D Systems) was injected on the GDF-11 coupled chip.

FIG. 5 A-204 Reporter Gene Assay. The figure shows the Reporter vector:pGL3(CAGA)12 (described in Dennler et al, 1998, EMBO 17: 3091-3100.) TheCAGA12 motif is present in TGF-Beta responsive genes (PAI-1 gene), sothis vector is of general use for factors signaling through Smad2 and 3.

FIG. 6 Caronte inhibits GDF-11 signaling in the A-204 Reporter GeneAssay. An ActRIIA-Fc (“IIA muG2a”) fusion also inhibits GDF-11signaling.

FIG. 7 Caronte does not inhibit Activin A in the A-204 Reporter GeneAssay. An ActRIIA-Fc fusion (“IIA muG2a”), as expected, does inhibitActivin A signaling.

FIG. 8 Cerberus and Caronte both inhibit GDF-8 signaling in the A-204Reporter Gene Assay.

FIG. 9 Human Coco-Fc inhibits GDF-11 signaling in a cell based assay.Conditioned medium from cells expressing human Coco-mFc was tested forits effects on A-204 reporter gene expression in the presence of GDF-11.

FIG. 10 Human Cerberus-Fc is degraded in human serum. Conditioned mediumfrom cells expressing human Cerberus-Fc was incubated overnight at 37deg. C. with varying amounts of human serum (percentages of serum addedare shown at top), and resolved by SDS-PAGE. Cerberus was detected byWestern blot with a primary antibody: biotinylated polyclonalanti-cerberus, and a secondary antibody: avidin-HRP. The left lane ismolecular weight standards. The major band, at roughly 70 kD isCerberus-Fc, which is completely degraded when incubated with 5% humanserum.

DETAILED DESCRIPTION I. Overview

Cerberus is expressed in the anterior endomesoderm (Bouwmeester et al.,Nature 382: 595-601, 1996; Piccolo et al., Nature 397: 707-10, 1999;Rodriguez et al., Nature 401: 243-51, 1999) during development. Caronte,a chick ortholog, is involved in left-right asymmetry in the chickembryo (Rodriguez, supra). Cerberus functions as a multivalent growthfactor antagonist in the extracellular space and inhibits signaling byBMP-4, nodal, and Wnt (Belo et al., Genesis 26: 265-70, 2000). MouseCerberus binds to BMP proteins and nodal via independent sites (Piccolo,supra), whereas the Xenopus Cerberus also binds Wnt proteins andinhibits their actions (Belo, supra). Cerberus has the unique propertyof inducing ectopic heads in the absence of trunk structures (Piccolo,supra). The expression of Cerberus during gastrulation is activated bynodal-related signals in endoderm and by Spemann-organizer factors(Yamamoto et al., Dev Biol 257: 190-204, 2003).

Orthologs for Cerberus can be found in Xenopus tropicalis and Fugurubripes, but are missing in invertebrates. In Fugu rubripes, there isonly one ortholog for Cerberus. All orthologous genes for Cerberus havetwo exons; the first eight amino acids of the cystine-knot domain areencoded by the 3′ end of the first exon and the remainder of the motifby the second exon. In some orthologs, a predicted proteolytic cleavagesite can be found upstream of the beginning of the cystine-knot domain.

Coco is another member of the Cerberus/Dan family of proteins thatinhibits Nodal signaling.

In part, the present invention invention provides Coco or Cerberusderivatives for inhibiting Nodal, GDF-11 and/or myostatin function. Incertain embodiments, the Coco and Cerberus derivatives inhibit Nodal,GDF-11 and/or myostatin function without substantially compromising BMP(such as BMP-4) signaling (e.g., does not substantially bind BMP-4 orother BMPs). The subject Cerberus derivatives may also be used toinhibit BMP (such as BMP-4) signaling.

Exemplary preparations of the subject invention include Cerberuspolypeptide derivatives, including the N-terminal truncated versions ofCerberus. These so-called “Cerberus derivatives” can be used to reducethe severity of a pathologic condition, which is characterized, at leastin part, by an abnormal amount, development or metabolic activity ofmuscle or adipose tissue in a subject. For instance, the pharmaceuticalpreparations of the present invention can be administered in an amounteffective to prevent, ameliorate or reduce the severity of a wastingdisorder, such as cachexia, anorexia, DMD syndrome, BMD syndrome, AIDSwasting syndrome, muscular dystrophies, neuromuscular diseases, motorneuron diseases, diseases of the neuromuscular junction, andinflammatory myopathies.

II. Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope an meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants/sequencevariants Such comparisons typically comprise alignments of polymersequences, e.g., using sequence alignment programs and/or algorithmsthat are well known in the art (for example, BLAST, FASTA and MEGALIGN,to name a few). The skilled artisan can readily appreciate that, in suchalignments, where a mutation contains a residue insertion or deletion,the sequence alignment will introduce a “gap” (typically represented bya dash, or “A”) in the polymer sequence not containing the inserted ordeleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other micleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al. Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) (melting temperature) of 55° C.,can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30%formamide, 5×SSC, 0.5% SDS).

Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SSC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SSC. SSC is 0.15M NaCl, 0.015M Na-citrate.

“High stringent condition” is well understood in the art to encompassconditions of hybridization which allow hybridization of structurallyrelated, but not structurally dissimilar, nucleic acids. The term“stringent” is a term of art which is understood by the skilled artisanto describe any of a number of alternative hybridization and washconditions which allow annealing of only highly complementary nucleicacids.

Exemplary high stringent hybridization conditions is equivalent to about20-27° C. below the melting temperature (T_(m)) of the DNA duplex formedin about 1M salt. Many equivalent procedures exist and several popularmolecular cloning manuals describe suitable conditions for stringenthybridization and, furthermore, provide formulas for calculating thelength of hybrids expected to be stable under these conditions (see e.g.Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6 or 13.3.6; or pages 9.47-9.57 of Sambrook, et al. (1989)Molecular Cloning, 2^(nd) ed., Cold Spring Harbor Press).

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m), for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of micleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.51). For hybridization with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.8). A minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; preferablyat least about 15 nucleotides; and more preferably the length is atleast about 20 nucleotides.

Unless specified, the term “standard hybridization conditions” refers toa T_(m) of about 55° C., and utilizes conditions as set forth above. Ina preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

Suitable hybridization conditions for oligonucleotides (e.g., foroligonucleotide probes or primers) are typically somewhat different thanfor full-length nucleic acids (e.g., full-length cDNA), because of theoligonucleotides' lower melting temperature. Because the meltingtemperature of oligonucleotides will depend on the length of theoligonucleotide sequences involved, suitable hybridization temperatureswill vary depending upon the oligonucleotide molecules used. Exemplarytemperatures may be 37° C. (for 14-base oligonucleotides), 48° C. (for17-base oligonucleotides), 55° C. (for 20-base oligonucleotides) and 60°C. (for 23-base oligonucleotides). Exemplary suitable hybridizationconditions for oligonucleotides include washing in 6×SSC, 0.05% sodiumpyrophosphate, or other conditions that afford equivalent levels ofhybridization.

“Polypeptide,” “peptide” or “protein” are used interchangeably todescribe a chain of amino acids that are linked together by chemicalbonds called “peptide bonds.” A protein or polypeptide, including anenzyme, may be a “native” or “wild-type,” meaning that it occurs innature; or it may be a “mutant,” “variant,” or “modified,” meaning thatit has been made, altered, derived, or is in some way different orchanged from a native protein or from another mutant.

As used herein, the terms “Cerberus/Dan/Gremlin superfamily protein” and“CDG protein” are both used to signify the protein family whichcomprises Cerberus, Coco and other related proteins.

“Cerberus or Cerberus-like protein” refers to mammalian Cerberus andCerberus-like proteins, such as the murine (NCBI RefSeq ID NP_034017) orhuman (NCBI RefSeq ID NP_005445) Cerberus proteins (also see SEQ ID NOs.2 and 8, respectively, of US 2002/0164682 A1, the entire contents ofwhich is incorporated herein by reference), and other proteins whichshare sequence homology to the highly conserved cysteine pattern of theC-terminal portion of the mammalian Cerberus proteins. Exemplary aminoacid sequences for Cerberus proteins include

Murine Cerberus protein (NCBI RefSeq ID NP_034017):

(SEQ ID NO: 4)  1 mhlllvqllv llplgkadlc vdgcqsqgsl sfpllergrr dlhvanheea edkpdlfvav 61 phlmgtslag egqrqrgkml srlgrfwkkp etefypprdv esdhvssgmq avtqpadgrk121 versplqeea krfwhrfmfr kgpafqgvil pikshevhwe tcrtvpfnqt iahedcqkvv181 vqnnlcfgkc ssirfpgega dahsfcshcs ptkfttvhlm lnctsptpvv kmvmqveecq241 cmvktergee rlllagsqgs fipglpaskt np

Human Cerberus protein (NCBI RefSeq ID NP_005445):

(SEQ ID NO: 5)  1 mhlllfqllv llplgkttrh qdgrqnqssl spvllprnqr elptgnheea eekpdlfvav 61 phlvatspag egqrgrekml srfgrfwkkp eremhpsrds dsepfppgtq sliqpidgmk121 meksplreea kkfwhhfmfr ktpasqgvil pikshevhwe tcrtvpfsqt ithegcekvv181 vqnnlcfgkc gsvhfpgaaq hshtscshcl pakfttmhlp lnctelssvi kvvmlveecq241 ckvktehedg hilhagsqds fipgvsa

The mouse and human Cerberus are as disclosed in US 2002/0164682 A1, asSEQ ID NOs. 1 and 7 (incorporated herein by reference).

NCBI RefSeq ID NM_005454.1 (human Cerberus mRNA).

(SEQ ID NO: 6)  1 atgcatctcc tcttatttca gctgctggta ctcctgcctc taggaaagac cacacggcac 61 caggatggcc gccagaatca gagttctctt tcccccgtac tcctgccaag gaatcaaaga121 gagcttccca caggcaacca tgaggaagct gaggagaagc cagatctgtt tgtcgcagtg181 ccacaccttg tagccaccag ccctgcaggg gaaggccaga ggcagagaga gaagatgctg241 tccagatttg gcaggttctg gaagaagcct gagagagaaa tgcatccatc cagggactca301 gatagtgagc ccttcccacc tgggacccag tccctcatcc agccgataga tggaatgaaa361 atggagaaat ctcctcttcg ggaagaagcc aagaaattct ggcaccactt catgttcaga421 aaaactccgg cttctcaggg ggtcatcttg cccatcaaaa gccatgaagt acattgggag481 acctgcagga cagtgccctt cagccagact ataacccacg aaggctgtga aaaagtagtt541 gttcagaaca acctttgctt tgggaaatgc gggtctgttc attttcctgg agccgcgcag601 cactcccata cctcctgctc tcactgtttg cctgccaagt tcaccacgat gcacttgcca661 ctgaactgca ctgaactttc ctccgtgatc aaggtggtga tgctggtgga ggagtgccag721 tgcaaggtga agacggagca tgaagatgga cacatcctac atgctggctc ccaggattcc781 tttatcccag gagtttcagc ttga 

NM_009887.1 (mouse Cerberus mRNA).

(SEQ ID NO: 7)   1 gggggggggg ggggtcagag ggagctttct tttaggcccg tccatctgtg aatctaacct  61 cagtttctgg gaatcaggaa gcatgcatct cctcttagtt cagctgcttg ttctcttgcc 121 tctggggaag gcagacctat gtgtggatgg ctgccagagt cagggctctt tatcctttcc 181 tctcctagaa aggggtcgca gagatctcca cgtggccaac cacgaggagg cagaagacaa 241 gccggatctg tttgtggccg tgccacacct catgggcacc agcctggctg gggaaggcca 301 gaggcagaga gggaagatgc tgtccaggct tggaagattc tggaagaaac ctgagaccga 361 attttacccc ccaagggatg tggaaagcga tcatgtctca tcggggatgc aggccgtgac 421 tcagccagca gatgggagga aagtggagag atcacctcta caggaggaag ccaagaggtt 481 ctggcatcgg ttcatgttca gaaagggccc ggcgttccag ggagtcatcc tgcccatcaa 541 aagccacgaa gtacactggg agacctgcag gactgtgccc ttcaaccaga ccattgccca 601 tgaagactgt caaaaagtcg ttgtccagaa caacctttgc tttggcaaat gcagttccat 661 tcgttttccc ggagaagggg cagatgccca cagcttctgc tcccactgct cgcccaccaa 721 attcaccacc gtgcacttga tgctgaactg caccagccca acccccgtgg tcaagatggt 781 gatgcaagta gaagagtgtc agtgcatggt gaagacggaa cgtggagagg agcgcctcct 841 actggctggt tcccagggtt ccttcatccc tggacttcca gcttcaaaaa caaacccatg 901 aattacctca acagaaagca aaacctcaac agaataagtg agggttattc aatctggaaa 961 tgttatgtga gttatataaa gatcagtgga aaatatcttt ctctctccct ctctccccctl021 ctctcttctc tctattttct ctctctctct ctctctctct ctctctctct ctctctctcal081 cacacacaca cacacacaca cacacacaca catgtttgtg tttagacagg gtcttatgtall41 ttctcagctg gcctcaaact cacaatgtgg ctggggatga ttttaaactc ctgatccaat1201 tcctgagtgc tgggattaca gacatgctcc ataanacata gctcccagaa ggatttttaa1261 aagagatttt gcatgtttca aagttgcctt tgagactcag aaatattttg atntattgaal321 tggccttgcc acagatgtgg gaggcagctt gcttggtggc ccaagtattt tttttttgttl381 cgttcagaat tctccacatg aagtttttac tgttggttat ctggcgttga agaaggaatal441 gtgaaggtac ttttaacagt ttacacgtgg aaggggctca ggcactagga accaacctttl501 tcccggaata tgaggaaaat acatgaacag tattagagtc acttgaggaa gttactaggal561 aacgccataa gtctccaagt acattgtgag tcattttgaa ggacaatcgt gtatatagacl621 gaaatcttct actcgtatgc ttttgaatct tctagcaagt taggtttcta tgtttgggctl681 tcttcctatt gtctaagagt atgtgtgaca aattcaacct gacaaatacc tcaatggcaal741 attctgaccc tg 

It is also expected that Cerberus related proteins also exist in otherspecies, including family members in Xenopus, and Drosophila, C.elegans, zebrafish, as well as in all manmnals, for example, rats, miceand humans. “Cerberus or Cerberus-like proteins” also includes variantsof the Cerberus proteins, such as allelic variants or variants inducedby mutagenesis or deletions, and fragments of Cerberus proteins whichvariants and fragments retain myostatin binding activity.“Cerberus-like” proteins is also used to signify the family of proteinssharing structural and/or functional similarity, including thoseproteins which are described further herein. Such proteins may haveamino acid sequences sharing significant sequence identity (e.g., atleast about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) with the human ormouse Cerberus proteins, over the full-length, or at least within themyostatin binding domain of the human or mouse Cerberus. Cerberus-likeproteins also include proteins that have amino acid sequences that areencoded by nucleic acid sequences that hybridize under stringentconditions with the coding sequences for human or mouse Cerberus,particularly that portion of the coding sequence for the myostatinbinding domain. A Cerberus derivative or variant sequence may or may notlack the N-terminal BMP binding domain.

“Coco or Coco-like protein” refers to mammalian Coco proteins andrelated homologs, such as the human Coco protein of GenBank Accession22749329, and other proteins which share sequence homology to the highlyconserved cysteine pattern of the C-terminal portion of the mammalianCoco proteins. An exemplary amino acid sequences for human Coco proteinis

(SEQ ID NO: 3)  1 MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSW 61 KAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL121 RNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI181 EGCHCSPKA 

The human Coco coding sequence is disclosed in GenBank Accession22749328 (incorporated herein by reference).

(SEQ ID NO: 8)   1 agtccggaca gacagacagg cagacagacg cacggacaag cagatgctcc ttggccagct  61 atccactctt ctgtgcctgc ttagcggggc cctgcctaca ggctcaggga ggcctgaacc 121 ccagtctcct cgacctcagt cctgggctgc agccaatcag acctgggctc tgggcccagg 181 ggccctgccc ccactggtgc cagcttctgc ccttgggagc tggaaggcct tcttgggcct 241 gcagaaagcc aggcagctgg ggatgggcag gctgcagcgt gggcaagacg aggtggctgc 301 tgtgactctg ccgctgaacc ctcaggaagt gatccagggg atgtgtaagg ctgtgccctt 361 cgttcaggtg ttctcccggc ccggctgctc agccatacgc ctccgaaatc atctgtgctt 421 tggtcattgc tcctctctct acatccctgg ctcggacccc accccactag tcctgtgcaa 481 cagctgtatg cctgctcgca agcgttgggc acccgtggtc ctgtggtgtc tcactggcag 541 ctcagcctcc cgtcgacggg tgaagatatc caccatgctg atcgaggggt gtcactgcag 601 cccaaaagca tgaactgagc atcgtggatg ggtgcacgga gacacgcacc ttggagaaat 661 gaggggagat ggaccaagaa agacgtggac ctggatgatg tactctgggt caagagacca 721 gggatgcagg gttaggcaga caggtcccca gagtcctcac cctgctcccc agacagtaga 781 cacagtgccc gtcctggagt tgcaccactg atagtcacag cacacaatga ttgacaactc 841 actttttttt ttttttttga gatggagtct cgctctgtcg cccaggctgg agtgcagtgg 901 cgcaatctca gctcactgca agctccacct cccgggttta tgccattctc ctgtctcagc 961 ctcccgagta gctgggacta caggcacccg ccaacacgcc cggctaattt ttcgtattttl021 tagtaaagac agggtttcac cgtgttagcc aggatggtct ctatctcctg acctcgtgatl081 ctgcctgcct tggccttatt attttttttt tttaaggaca gagtctctct ctgtcacccall41 ggctggagtg caatggcgcg atcttggctc actgtaactt ccacttgcca ggctcaagca1201 gttctcctgc ctcagcctcc tgagtagctg ggactacagg cacccgccac catgcccagc1261 taatttttgt atttttagta gagacagagt ttcaccatat tagcctggct ggtctcaaacl321 tcctggcctc aggtgatctg cccacctcgg cctcccaaag tgctgggatc aaatccactgl381 ttaatcatta ggctgaactg tctcttatag aatgaggtca aagacactcc cagttgcaggl441 gagggtagat ggccccaccc agaccgagag acacagtgat gacctcagcc tagggacaccl501 aaaaaaaaaa aaaaaaaaaa cccaaaccaa aaacgcaaac caaagcaggc aggcagacagl561 ctgctggggg aaatcctggg gtccttgaga cagaggcagg accctcgtgt tcccagctgcl621 ctcttgcctt gatagtggtg ctgtgtccct ctcagacccc ccacctgagt ctccacagagl681 ccccacgcct ggcatggcat tccacagaaa ccataaaggt tggctgagtc c 

It is also expected that Coco-related proteins also exist in otherspecies, including family members in Xenopus, and Drosophila, C.elegans, zebrafish, as well as in all manmnals, for example, rats, miceand non-human primates. “Coco or Coco-like proteins” also includesvariants of the naturally occurring Coco proteins, such as allelicvariants or variants induced by mutagenesis or deletions, and fragmentsof Coco proteins which variants and fragments retain myostatin bindingactivity. “Coco-like” proteins is also used to signify the family ofproteins sharing structural and/or functional similarity, includingthose proteins which are described further herein. Such proteins mayhave amino acid sequences sharing significant sequence identity (e.g.,at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) with the humanCoco protein, over the full-length, or at least within the myostatinbinding domain of the human Coco. Coco-like proteins also includeproteins that have amino acid sequences that are encoded by nucleic acidsequences that hybridize under stringent conditions with the codingsequences for human Coco, particularly that portion of the codingsequence for the myostatin binding domain. A Coco derivative or variantsequence may or may not lack the N-terminal BMP binding domain.

Unless specifically stated otherwise, “Cerberus (derivative)therapeutics” or its grammatical variations include the full-length orthe N-terminally truncated versions of Cerberus therapeutics.

As used herein, the term “Cerberus or Cerberus-like activity” refers toone or more of the activities which are exhibited by the mammalianCerberus-like proteins of the present invention. In particular,“Cerberus or Cerberus-like activity” includes the ability to induce,enhance and/or inhibit the formation, growth, proliferation,differentiation, maintenance of neurons and/or related neural cells andtissues such as brain cells, Schwann cells, glial cells and astrocytes.“Cerberus or Cerberus-like” activity also includes the ability to inducemolecular markers of neuroendocrine or ectoderm tissue, such as OTX2,N-CAM, MASH, chromagranin, and AP2, as well as the ability to induce theformation of neurons and/or related neural cells and tissues such asbrain cells, Schwann cells, glial cells and astrocytes. “Cerberus orCerberus-like activity” may also include the ability to regulate theinteraction of ligands and their protein receptors. “Cerberus orCerberus-like activity” may further include the ability to regulate theformation, differentiation, proliferation and/or maintenance of othercells and/or tissue, for example connective tissue, organs and woundhealing. In particular, “Cerberus or Cerberus-like activity” may includethe ability to enhance and/or inhibit the formation, growth,proliferation, differentiation and/or maintenance of cardiac, spleen,liver, pancreas, stomach, kidney, lung and brain cells and tissue, aswell as osteoblasts and bone, chondrocytes and cartilage, tendon,epidermis and muscle. “Cerberus and Cerberus-like activity” alsoincludes the activities of Cerberus and Cerberus-like protein in theassays described in the examples and specification herein.

Cerberus and Cerberus-like nucleotide sequences in mouse and human areas disclosed in US 2002/0164682 A1, as SEQ ID NOs: 1 and 7 (incorporatedherein by reference). Also see NCBI RefSeq ID NM_005454.1 (human) andNM_009887.1 (mouse).

In certain related embodiments, the mysotatin inhibitor is a polypeptidethat includes a myostatin binding domain of a Coco protein, such as thehuman Coco protein shown in FIG. 3 or in GenBank Accession number22749329. An exemplary human Coco protein sequence is

(SEQ ID NO: 3)  1 MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSW 61 KAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL121 RNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI181 EGCHCSPKA 

The terms “antibody” and “antibody agent” are used interchangeablyherein, and refer to an immunoglobulin molecule obtained by in vitro orin vivo generation of the humoral response, and includes both polyclonaland monoclonal antibodies. The term also includes genetically engineeredforms such as chimeric antibodies (e.g., humanized murine antibodies),heteroconjugate antibodies (e.g., bispecific antibodies), andrecombinant single chain Fv fragments (scFv). The term “antibody” alsoincludes antigen binding forms of antibodies (e.g., Fab′, F(ab′)₂, Fab,Fv, rIgG, and, inverted IgG).

The term “antigen binding fragment” includes any portion of an antibodythat binds to a target epitope. An antigen binding fragment may be, forexample, a polypeptide including a CDR3 region, or other fragment of animmunoglobulin molecule which retains the affinity and specificity ofthe myostatin epitope.

“Specifically binds” includes reference to the preferential associationof a ligand, in whole or part, with a particular target molecule (i.e.,“binding partner” or “binding moiety”) relative to compositions lackingthat target molecule. It is, of course, recognized that a certain degreeof non-specific interaction may occur between the subject myostatinneutralizing antibodies and a other proteins. Nevertheless, specificbinding, may be distinguished as mediated through specific recognitionof the myostatin protein. Typically specific binding results in a muchstronger association between the antibody and myostatin protein thanbetween the antibody and other proteins, e.g., GDF11. Specific bindingby an antibody to myostatin under such conditions requires an antibodythat is selected for its specificity for a particular protein. Theaffinity constant (Ka, as opposed to Kd) of the antibody binding sitefor its cognate monovalent antigen is at least 10⁷, usually at least10⁸, preferably at least 10⁹, more preferably at least 10¹⁰, and mostpreferably at least 10¹¹M. A variety of immunoassay formats areappropriate for selecting antibodies specifically reactive withmyostatin. For example, solid-phase ELISA immunoassays are routinelyused to select monoclonal antibodies specifically reactive with aprotein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual,Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions that can be used to determinespecific reactivity.

Immunoassays in the competitive binding format can be used to determinecross-reactivity of antibodies with myostatin, e.g., to identify whethera test antibody is a myostatin neutralizing antibody. For example, themyostatin protein, or a fragment thereof is immobilized to a solidsupport. Test antibodies are added to the assay compete with the bindingof a TGF receptor, such as ActRII or ALK7, to the immobilized antigen.The ability of the test antibodies to compete with the binding of a TGFreceptor to the immobilized myostatin antigen is compared.

Similarly, immunoassays in the competitive binding format can be used todetermine cross-reactivity determinations, e.g., to determine thespecificity of a myostatin neutralizing antibody. For example, themyostatin protein, or the myostatin epitope thereof is immobilized to asolid support. Epitopes from other proteins, such as GDF-11, Nodal orBMP-4 or other proteins having sequence homology with myostatin areadded to the assay to compete with the binding of a potential myostatinneutralizing antibody to the immobilized antigen. The ability of thetest peptides to compete with the binding of potential myostatinneutralizing antibody with the immobilized myostatin antigen iscompared. The percent cross-reactivity of the potential myostatinneutralizing antibody for the other antigens is calculated, usingstandard calculations. In certain preferred embodiments, the subjectmyostatin neutralizing antibodies have less than 10% cross-reactivitywith GDF-11. In other preferred embodiments, the subject myostatinneutralizing antibodies have less than 1%, 5%, or 10% cross-reactivitywith BMP-4.

III. Exemplary Cerberus and Coco Derivatives

In certain embodiments, the mysotatin inhibitor is a Cerberuspolypeptide sharing at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% ormore sequence identity over the full-length of the human or mouseCerberus protein (infra).

In certain other embodiments, the mysotatin inhibitor is a polypeptidethat includes a Cerberus sequence obtained from human, mouse, or otherspecies, their variants or derivatives, including N-terminally truncatedversions of Cerberus. The full-length mouse and human Cerberus proteins,disclosed as SEQ ID NOs: 2 and 8, respectively, in US 2002/0164682 A1,are also disclosed in NCBI RefSeq format below:

Human Cerberus full length protein:

(SEQ ID NO: 5)  1 MHLLLFQLLV LLPLGKTTRH QDGRQNQSSL SPVLLPRNQR ELPTGNHEEA EEKPDLFVAV 61 PHLVATSPAG EGQRQREKML SRFGRFWKKP EREMHPSRDS DSEPFPPGTQ SLIQPIDGMK121 MEKSPLREEA KKFWHHFMFR KTPASQGVIL PIKSHEVHWE TCRTVPFSQT ITHEGCEKVV181 VQNNLCFGKC GSVHFPGAAQ HSHTSCSHCL PAKFTTMHLP LNCTELSSVI KVVMLVEECQ24l CKVKTEHEDG HILHAGSQDS FIPGVSA

Residues 106-119 (from any one of which residues the subject Cerberusderivatives may begin), and residues 241-267 (to any one of whichresidues the subject Cerberus derivatives may end) are underlined.

Mouse Cerberus full length protein:

(SEQ ID NO: 4)  1 MHLLLVQLLV LLPLGKADLC VDGCQSQGSL SFPLLERGRR DLHVANHEEA EDKPDLFVAV 61 PHLMGTSLAG EGQRQRGKML SRLGRFWKKP ETEFYPPRDV ESDHVSSGMQ AVTQPADGRK121 VERSPLQEEA KRFWHRFMFR KGPAFQGVIL PIKSHEVHWE TCRTVPFNQT IAHEDCQKVV181 VQNNLCFGKC SSIRFPGEGA DAHSFCSHCS PTKFTTVHLM LNCTSPTPVV KMVMQVEECQ241 CMVKTERGEE RLLLAGSQGS FIPGLPASKT NP

Residues 106-119 (from any one of which residues the subject Cerberusderivatives may begin), and residues 241-272 (to any one of whichresidues the subject Cerberus derivatives may end) are underlined. Notethat the mouse protein is largely homologous to the human proteinthroughout the sequences, with the exception of 5 additional residues atthe C-terminus. Therefore, whenever a non-human Cerberus derivative isused, the residue numbers refers to those corresponding to the humansequences.

As described above, in certain embodiments, preferred fragments of thehuman Cerberus derivative proteins are ones which begins anywhere fromresidues 106-119 (inclusive) at the N-terminus, and ends anywhere afterresidue 241.

Also included are Cerberus derived variant sequence, including mutantsor variants of the wild-type myostatin binding domains that retainmyostatin binding activity, optionally substantially loses BMP-4binding. Variant sequences without BMP binding affinity may be desirableas a way to alter selectivity of the inhibitor (e.g., relative to GDF-11or nodal binding, where preferential binding to one of the proteinsoccur. Also includes more preferential—higher affinity thanwild-type—binding to myostatin, or more discrimitory—lower affinity thanwild-type truncated version—binding to BMP-4), alter other bindingcharacteristics with respect to myostatin (such as K_(d), and/or K_(on)or K_(off) rates), or improve biodistribution or half life in vivo or onthe shelf.

Certain other Cerberus sequences are listed below based on homologysearch in databases of identified proteins, and the subject variantCerberus polypeptides can be derived from those proteins as well. Sincethese sequences are retrieved from public databases available on theinternet, additional homologs of the proteins in other species may beobtained as these databases are being updated. Furthermore, otherspecies of Cerberus proteins, especially those of mammals, can bereadily obtained by standard molecular biology protocols, such as PCR,low stringency hybridization, Ab-mediated screening of expressionlibraries using antibodies cross-reacting with identified Cerberushomologs in target species, etc.

For example, sequence alignments using softwares such as DNAStar'sMegaAlign (supra) can identify the most conserved regions in the knownmembers of a protein family. PCR can then be carried out usingdegenerate oligoes covering such most conserved regions, and templatesDNA from the target organism. In preferred embodiments, such conservedregions include the kinase domain, and/or the ligand binding domain.

These same conserved regions may be used to generate probes forscreening nucleic acid libraries at moderate to low stringencyhybridization conditions (see definition section).

Xenopus homolog: gi|1513088.

Fugu rubripes: FuguGenscan_32561/SINFRUP00000076662.

In certain embodiments, the mysotatin inhibitor is a Cerberuspolypeptide sharing at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% ormore sequence identity over the full-length of the human or mouseCerberus protein (infra).

In certain other embodiments, the mysotatin inhibitor is a polypeptidethat includes a Coco sequence obtained from human, mouse, or otherspecies, their variants or derivatives, including N-terminally truncatedversions of Coco. The full-length human Coco protein is disclosed above.

The various Cerberus and Coco polypeptides may be prepared as fusionproteins. A fusion protein may include one or more additionalpolypeptide portion that enhance one or more of in vivo stability, invivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. Forexample, a fusion protein may include an immunoglobulin Fc domain and/ora purification subsequence selected from: an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. The myostatin antagonistprotein may include one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent.

A fusion protein or coupled protein system (e.g. non-fusion covalentlinkage by crosslinking) may also include a second myostatin inhibitordomain, which is a polypeptide affinity reagent that selectively bindsto myostatin and competes with the binding of an ALK7 or ALK4 receptor.The affinity reagent may be an antibody agent. An antibody agent may be,for example, a recombinant antibody; a monoclonal antibody; a VH domain;a VL domain; an scFv; an Fab fragment; an Fab′ fragment; an F(ab′)2; anFv; or a disulfide linked Fv, a fully human antibody or a humanizedchimeric antibody, or an antigen binding fragment thereof. An affinityreagent is a peptide or scaffolded peptide that selectively binds tomyostatin and competes with the binding of an ALK7 or ALK4 receptor. Anaffinity reagent may include a myostatin binding domain of ALK7 or ALK4.For example, an extracellular domain of ALK7 or ALK4 (preferably humanALK7 or ALK4) may be used. The affinity reagent may be a small organicmolecule that selectively binds to myostatin and competes with thebinding of an ALK7 or ALK4 receptor.

An example of a human ALK7 myostatin binding domain is shown below:

(SEQ ID NO: 9) LKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLP 

An example of a human ALK4 myostatin binding domain is shown below:

(SEQ ID NO: 10) ALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDY 

As shown herein, Caronte, and therefore human Cerberus and presumablyCoco also, does not substantially inhibit Activin A signaling in an A204Reporter Gene Assay. Thus, such myostatin antagonists will preferablyexhibit little or no interaction with Activin A-mediated signaling.

IV. Examplary Therapeutic Uses

The subject variant Coco and Cerberus polypeptides, such as thefull-length and the N-terminally truncated Cerberus derivatives or Cocoderivatives, can be used in a number of therapeutic settings to treat anumber of diseases resulting from or exacerbated by the presence ofmyostatin.

In certain embodiments, the subject polypeptides and derivatives thereofare used as part of a treatment for a muscular dystrophy. The term“muscular dystrophy” refers to a group of degenerative muscle diseasescharacterized by gradual weakening and deterioration of skeletal musclesand sometimes the heart and respiratory muscles. Muscular dystrophiesare genetic disorders characterized by progressive muscle wasting andweakness that begin with microscopic changes in the muscle. As musclesdegenerate over time, the person's muscle strength declines. Exemplarymuscular dystrophies that can be treated with a regimen including thesubject myostatin include: Duchenne Muscular Dystrophy (DMD), BeckerMuscular Dystrophy (BMD), Emery-Dreifuss Muscular Dystrophy (EDMD),Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulohumeral MuscularDystrophy (FSH or FSHD) (Also known as Landouzy-Dejerine), MyotonicDystrophy (MMD) (Also known as Steinert's Disease), OculopharyngealMuscular Dystrophy (OPMD), Distal Muscular Dystrophy (DD), CongenitalMuscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

In DMD, boys begin to show signs of muscle weakness as early as age 3.The disease gradually weakens the skeletal or voluntary muscles, thosein the arms, legs and trunk. By the early teens or even earlier, theboy's heart and respiratory muscles may also be affected. BMD is a muchmilder version of DMD. Its onset is usually in the teens or earlyadulthood, and the course is slower and far less predictable than thatof DMD. (Though DMD and BMD affect boys almost exclusively, in rarecases they can affect girls.

Until the 1980s, little was known about the cause of any kind ofmuscular dystrophy. In 1986, the dystrophin gene deficiency wasidentified as the cause of DMD. BMD results from different mutations inthe same gene. BMD patients have some dystrophin, but it's eitherinsufficient in quantity or poor in quality. Having some dystrophinprotects the muscles of those with BMD from degenerating as badly or asquickly as those of people with DMD.

Recent researches demonstrate that blocking or eliminating Myostatinfunction in vivo can effectively treat at least certain symptoms in DMDand BMD patients (Bogdanovich et al., supra; Wagner et al., supra).Thus, the subject Cerberus derivatives, especially the N-terminallytruncated versions thereof, constitute an alternative means of blockingthe function of Myostatin in vivo in DMD and BMD patients.

Similarly, the subject Coco or Cerberus derivatives, especially theN-terminally truncated versions thereof, provide an effective means toincrease muscle mass in other disease conditions that are in need ofmuscle growth. For example, Gonzalez-Cadavid et al. (supra) reportedthat that Myostatin expression correlates inversely with fat-free massin humans and that increased expression of the Myostatin gene isassociated with weight loss in men with AIDS wasting syndrome. Byinhibiting the function of Myostatin in AIDS patients, at least certainsymptoms of AIDS may be alleviated, if not completely eliminated, thussignificantly improving quality of life in AIDS patients.

Since loss of Myostatin function is also associated with fat losswithout diminution of nutrient intake (Zimmers et al., supra; McPherronand Lee, supra), the subject Coco or Cerberus derivatives, especiallythe N-terminally truncated versions thereof, may further be used as atherapeutic agent for slowing or preventing the development of obesityand type II diabetes.

The cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. Progressive weight loss in canceranorexia-cachexia syndrome is a common feature of many types of cancerand is responsible not only for a poor quality of life and poor responseto chemotherapy, but also a shorter survival time than is found inpatients with comparable tumors without weight loss. Associated withanorexia, fat and muscle tissue wasting, psychological distress, and alower quality of life, cachexia arises from a complex interactionbetween the cancer and the host. It is one of the most common causes ofdeath among cancer patients and is present in 80% at death. It is acomplex example of metabolic chaos effecting protein, carbohydrate, andfat metabolism. Tumors produce both direct and indirect abnormalities,resulting in anorexia and weight loss. Currently, there is no treatmentto control or reverse the process.

Cancer anorexia-cachexia syndrome affects cytokine production, releaseof lipid-mobilizing and proteolysis-inducing factors, and alterations inintermediary metabolism. Although anorexia is common, a decreased foodintake alone is unable to account for the changes in body compositionseen in cancer patients, and increasing nutrient intake is unable toreverse the wasting syndrome. Cachexia should be suspected in patientswith cancer if an involuntary weight loss of greater than five percentof premorbid weight occurs within a six-month period.

Since systemic overexpression of Myostatin in adult mice was found toinduce profound muscle and fat loss analogous to that seen in humancachexia syndromes (Zimmers et al., supra), the subject Coco or Cerberusderivatives, especially the N-terminally truncated versions thereof as apharmaceutical composition can be beneficially used as a Myostatinantagonist/blocker to prevent, treat, or alleviate the symptoms of thecachexia syndrome, where muscle growth is desired.

In certain embodiments, the subject variant Coco or Cerberuspolypeptides, particularly the N-terminally truncated Cerberusderivatives, can be used to form pharmaceutical compositions that can bebeneficially used to prevent, treat, or alleviate symptoms of a host ofdiseases involving neurodegeneration. While not wishing to be bound byany particular theory, the subject Cerberus derivatives may antagonizethe inhibitory feedback mechanism mediated through the wild-type ALK7receptor, thus allowing new neuronal growth and differentiation. Thesubject Cerberus derivative as a pharmaceutical composition can bebeneficially used to prevent, treat, or alleviate symptoms of diseaseswith neurodegeneration, including Alzheimer's Disease (AD), Parkinson'sDisease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease,etc.

Alzheimer's disease (AD) is a chronic, incurable, and unstoppablecentral nervous system (CNS) disorder that occurs gradually, resultingin memory loss, unusual behavior, personality changes, and a decline inthinking abilities. These losses are related to the death of specifictypes of brain cells and the breakdown of connections between them.

AD has been described as childhood development in reverse. In mostpeople with AD, symptoms appear after the age 60. The earliest symptomsinclude loss of recent memory, faulty judgment, and changes inpersonality. Later in the disease, those with AD may forget how to dosimple tasks like washing their hands. Eventually people with AD loseall reasoning abilities and become dependent on other people for theireveryday care. Finally, the disease becomes so debilitating thatpatients are bedridden and typically develop coexisting illnesses. ADpatients most commonly die from pneumonia, 8 to 20 years from diseaseonset.

Parkinson's disease (PD) is a chronic, incurable, and unstoppable CNSdisorder that occurs gradually and results in uncontrolled bodymovements, rigidity, tremor, and gait difficulties. These motor systemproblems are related to the death of brain cells in an area of the brainthat produces dopamine—a chemical that helps control muscle activity.

In most people with PD, symptoms appear after age 50. The initialsymptoms of PD are a pronounced tremor affecting the extremities,notably in the hands or lips. Subsequent characteristic symptoms of PDare stiffness or slowness of movement, a shuffling walk, stoopedposture, and impaired balance. There are wide ranging secondary symptomssuch as memory loss, dementia, depression, emotional changes, swallowingdifficulties, abnormal speech, sexual dysfunction, and bladder and bowelproblems. These symptoms will begin to interfere with routineactivities, such as holding a fork or reading a newspaper. Finally,people with PD become so profoundly disabled that they are bedridden.People with PD usually die from pneumonia.

Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neurondisease) is a chronic, incurable, and unstoppable CNS disorder thatattacks the motor neurons, components of the CNS that connect the brainto the skeletal muscles. In ALS, the motor neurons deteriorate andeventually die, and though a person's brain normally remains fullyfunctioning and alert, the command to move never reaches the muscles.

Most people who get ALS are between 40 and 70 years old. The first motorneurons that weaken are those leading to the arms or legs. Those withALS may have trouble walking, they may drop things, fall, slur theirspeech, and laugh or cry uncontrollably. Eventually the muscles in thelimbs begin to atrophy from disuse. This muscle weakness will becomedebilitating and a person will need a wheel chair or become unable tofunction out of bed. Most ALS patients die from respiratory failure orfrom complications of ventilator assistance like pneumonia, 3-5 yearsfrom disease onset.

The causes of these neurological diseases has remained largely unknown.They are conventionally defined as distinct diseases, yet clearly showextraordinary similarities in basic processes and commonly demonstrateoverlapping symptoms far greater than would be expected by chance alone.Current disease definitions fail to properly deal with the issue ofoverlap and a new classification of the neurodegenerative disorders hasbeen called for.

Huntington's disease (HD) is another neurodegenerative disease resultingfrom genetically programmed degeneration of neurons in certain areas ofthe brain. This degeneration causes uncontrolled movements, loss ofintellectual faculties, and emotional disturbance. HD is a familialdisease, passed from parent to child through a dominant mutation in thewild-type gene. Some early symptoms of HD are mood swings, depression,irritability or trouble driving, learning new things, remembering afact, or making a decision. As the disease progresses, concentration onintellectual tasks becomes increasingly difficult and the patient mayhave difficulty feeding himself or herself and swallowing. The rate ofdisease progression and the age of onset vary from person to person.

Tay-Sachs disease and Sandhoff disease are glycolipid storage diseasescaused by the lack of lysosomal β-hexosaminidase (Gravel et al., in TheMetabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill,New York, pp. 2839-2879, 1995). In both disorders, G_(M2) gangliosideand related glycolipidssubstrates for β-hexosaminidaseaccumulate in thenervous system and trigger acute neurodegeneration. In the most severeforms, the onset of symptoms begins in early infancy. A precipitousneurodegenerative course then ensues, with affected infants exhibitingmotor dysfunction, seizure, visual loss, and deafness. Death usuallyoccurs by 2-5 years of age. Neuronal loss through an apoptotic mechanismhas been demonstrated (Huang et al., Hum. Mol. Genet. 6: 1879-1885,1997).

It is well-known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease. Shi etal. (J. Clin. Invest. 98: 1979-1990, 1996) examined apoptosis induced byHIV-1 infection of the central nervous system (CNS) in an in vitro modeland in brain tissue from AIDS patients, and found that HIV-1 infectionof primary brain cultures induced apoptosis in neurons and astrocytes invitro. Apoptosis of neurons and astrocytes was also detected in braintissue from 10/11 AIDS patients, including 5/5 patients with HIV-1dementia and 4/5 nondemented patients.

Neuronal loss is a also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats.

The subject Cerberus derivatives, including the N-terminally truncatedCerberus derivatives are also useful to prevent, treat, and alleviatesymptoms of various PNS disorders, such as the ones described below. ThePNS is composed of the nerves that lead to or branch off from the CNS.The peripheral nerves handle a diverse array of functions in the body,including sensory, motor, and autonomic functions. When an individualhas a peripheral neuropathy, nerves of the PNS have been damaged. Nervedamage can arise from a number of causes, such as disease, physicalinjury, poisoning, or malnutrition. These agents may affect eitherafferent or efferent nerves. Depending on the cause of damage, the nervecell axon, its protective myelin sheath, or both may be injured ordestroyed.

The term peripheral neuropathy encompasses a wide range of disorders inwhich the nerves outside of the brain and spinal cord—peripheralnerves—have been damaged. Peripheral neuropathy may also be referred toas peripheral neuritis, or if many nerves are involved, the termspolyneuropathy or polyneuritis may be used.

Peripheral neuropathy is a widespread disorder, and there are manyunderlying causes. Some of these causes are common, such as diabetes,and others are extremely rare, such as acrylamide poisoning and certaininherited disorders. The most common worldwide cause of peripheralneuropathy is leprosy. Leprosy is caused by the bacterium Mycobacteriumleprae, which attacks the peripheral nerves of affected people.According to statistics gathered by the World Health Organization, anestimated 1.15 million people have leprosy worldwide.

Leprosy is extremely rare in the United States, where diabetes is themost commonly known cause of peripheral neuropathy. It has beenestimated that more than 17 million people in the United States andEurope have diabetes-related polyneuropathy. Many neuropathies areidiopathic—no known cause can be found. The most common of the inheritedperipheral neuropathies in the United States is Charcot-Marie-Toothdisease, which affects approximately 125,000 persons.

Another of the better known peripheral neuropathies is Guillain-Barrésyndrome, which arises from complications associated with viralillnesses, such as cytomegalovirus, Epstein-Barr virus, and humanimmunodeficiency virus (HIV), or bacterial infection, includingCampylobacter jejuni and Lyme disease. The worldwide incidence rate isapproximately 1.7 cases per 100,000 people annually. Other well-knowncauses of peripheral neuropathies include chronic alcoholism, infectionof the varicella-zoster virus, botulism, and poliomyelitis. Peripheralneuropathy may develop as a primary symptom, or it may be due to anotherdisease. For example, peripheral neuropathy is only one symptom ofdiseases such as amyloid neuropathy, certain cancers, or inheritedneurologic disorders. Such diseases may affect the peripheral nervoussystem (PNS) and the central nervous system (CNS), as well as other bodytissues.

Other PNS diseases treatable with the subject Cerberus derivatives,especially the N-terminally truncated Cerberus derivatives include:Brachial Plexus Neuropathies (Diseases of the cervical and firstthoracic roots, nerve trunks, cords, and peripheral nerve components ofthe brachial plexus. Clinical manifestations include regional pain,paresthesia; muscle weakness, and decreased sensation in the upperextremity. These disorders may be associated with trauma, includingbirth injuries; thoracic outlet syndrome; neoplasms, neuritis,radiotherapy; and other conditions. See Adams et al., Principles ofNeurology, 6^(th) ed, pp1351-2); Diabetic Neuropathies (Peripheral,autonomic, and cranial nerve disorders that are associated with disbetesmellitus. These conditions usually result from diabetic microvascularinjury involving small blood vessels that supply nerves (vasa nervorum).Relatively common conditions which may be associated with diabeticneuropathy include third nerve palsy; mononeuropathy; mononeuropathymultiplex; diabetic amyotrophy; a painful polyneuropathy; autonomicneuropathy; and thoracoabdominal neuropathy. See Adams et al.,Principles of Neurology, 6^(th) ed, p1325); Mononeuropathies (Disease ortrauma involving a single peripheral nerve in isolation, or out ofproportion to evidence of diffuse peripheral nerve dysfunction.Mononeuropathy multiplex refers to a condition characterized by multipleisolated nerve injuries. Mononeuropathies may result from a wide varietyof causes, including ischemia; traumatic injury; compression; connectivetissue diseases; cumulative trauma disorders; and other conditions);Neuralgia (Intense or aching pain that occurs along the course ordistribution of a peripheral or cranial nerve); Peripheral NervousSystem Neoplasms (Neoplasms which arise from peripheral nerve tissue.This includes neurofibromas; Schwannomas; granular cell tumors; andmalignant peripheral nerve sheath tumors. See DeVita Jr et al., Cancer:Principles and Practice of Oncology, 5^(th) ed, pp1750-1); NerveCompression Syndromes (Mechanical compression of nerves or nerve rootsfrom internal or external causes. These may result in a conduction blockto nerve impulses, due to, for example, myelin sheath dysfunction, oraxonal loss. The nerve and nerve sheath injuries may be caused byischemia; inflammation; or a direct mechanical effect); Neuritis (Ageneral term indicating inflammation of a peripheral or cranial nerve.Clinical manifestation may include pain; paresthesias; paresis; orhyperthesia); Polyneuropathies (Diseases of multiple peripheral nerves.The various forms are categorized by the type of nerve affected (e.g.,sensory, motor, or autonomic), by the distribution of nerve injury(e.g., distal vs. proximal), by nerve component primarily affected(e.g., demyelinating vs. axonal), by etiology, or by pattern ofinheritance).

In certain embodiments, the subject full-length Coco or Cerberuspolypepetides or variants thereof are used as part of a treatment fordiseases or conditions characterized by excessive or undesirable levelsof BMP, such as the ones described below.

The heterotopic ossification of muscles, tendons, and ligaments is acommon problem faced by orthopaedic surgeons. Hannallah et al. (J BoneJoint Surg Am. 2004 January; 86-A(1):80-91) investigated the ability ofNoggin (a BMP [bone morphogenetic protein] antagonist) to inhibitheterotopic ossification. Three varying doses of Noggin-expressingmuscle-derived stem cells inhibited the heterotopic ossificationelicited by BMP-4-expressing muscle-derived stem cells. Each of threevarying doses of Noggin-expressing muscle-derived stem cells alsosignificantly inhibited the heterotopic ossification elicited bydemineralized bone matrix. All eleven animals that underwent Achillestenotomy developed heterotopic ossification at the site of the injury inthe control limbs. In contrast, the limbs treated with theNoggin-expressing muscle-derived stem cells had a reduction in theformation of heterotopic ossification of 83% and eight of the elevenanimals had no radiographic evidence of heterotopic ossification(p<0.05). Thus, delivery of Noggin mediated by muscle-derived stem cellscan inhibit heterotopic ossification caused by BMP-4, demineralized bonematrix, and trauma in an animal model, indicating that gene therapy todeliver BMP inhibitors (Noggin or Cerberus) may become a powerful methodto inhibit heterotopic ossification in targeted areas of the body. Seealso Glaser et al. (J Bone Joint Surg Am. 2003 December;85-A(12):2332-42).

Osteoarthritis (OA) is a joint disease characterized by osteophytedevelopment, fibrosis, and articular cartilage damage. Effects ofexogenous transforming growth factor beta (TGFbeta) isoforms and bonemorphogenetic proteins (BMPs) suggest a role for these growth factors inthe pathogenesis of OA. Scharstuhl et al. (Arthritis Rheum. 2003December; 48(12):3442-51) used adenoviral overexpression of TGF-beta andBMP antagonists to block the signaling of TGF-beta and BMP. Theinhibitors studied include a secreted, pan-specific TGF-beta antagonistcalled murine latency-associated peptide 1 (mLAP-1), intracellularinhibitory Smad6 (a BMP antagonist), and Smad7 (a TGF-beta/BMPinhibitor). Intraarticular injection of papain caused increased proteinexpression of several TGF-beta and BMP isoforms in synovium andcartilage. Adenovirus transfection into the joint resulted in a strongexpression of the transgenes in the synovial lining. Overexpression ofmLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyteformation compared with that in controls. Smad6 and Smad7 overexpressionalso significantly decreased synovial thickening. Furthermore, thesecreted TGF-beta inhibitor mLAP-1 increased articular cartilage PGloss. These results indicate a pivotal role of excessive endogenousTGF-beta and BMP in the development of osteophytes and synovialthickening, implicating excessive endogenous TGFbeta and BMP in thepathogenesis of OA. In contrast, the prevention of cartilage damage byendogenous TGF-beta signifies the protective role of TGF-beta inarticular cartilage. Thus the subject Coco or Cerberus pharmaceuticalcompositions can be used as BMP antagonists to treat OA, including thedevelopment of osteophytes and synovial thickening.

In an analysis of normal ovarian surface epithelium (OSE) and ovariancancer (OC) cells, Shepherd and Nachtigal (Endocrinology. 2003 August;144(8):3306-14) observed BMP4 mRNA expression and found that primary OCcells produce mature BMP4. In addition, each member of the downstreamsignaling pathway was expressed in primary OSE and OC cells. Smad1 wasphosphorylated and underwent nuclear translocation in normal OSE and OCcells upon treatment with BMP4. Interestingly, the BMP target genes ID1and ID3 were up-regulated 10- to 15-fold in primary OC cells, comparedwith a 2- to 3-fold increase in normal OSE. The growth of severalprimary OC cells was relatively unaltered by BMP4 treatment; however,long-term BMP4 treatment of primary OC cells resulted in decreased celldensity as well as increased cell spreading and adherence. These datademonstrate the existence and putative function of BMP signaling innormal OSE and OC cells, and thus the subject Cerberus pharmaceuticalpreparations can be used to regulate BMP4 signaling in OC pathogenesis.

Fibrodysplasia ossificans progressiva (FOP), a rare genetic disablingdisease characterized by heterotopic bone formation, is of specialinterest for general medicine since the bone morphogenetic proteins(especially BMP-4) involved in its pathogenesis are known to play a rolein skeletal morphogenesis, and the gene antagonist to BMP-4 (such asnoggin) might be useful in preventing lamellar bone formation. SeeBlaszczyk et al. (Eur J Dermatol. 2003 May-Jim; 13(3):234-7). Thus thesubject Cerberus therapeutics may also be used to treat FOP.

Atherosclerosis is now viewed as an inflammatory disease occurringpreferentially in arterial regions exposed to disturbed flow conditions,including oscillatory shear stress (OS), in branched arteries. Sorescuet al. (J Biol Chem. 278(33):31128-35, 2003) suggest that BMP4 is amechanosensitive, inflammatory factor playing a critical role in earlysteps of atherogenesis in the lesion-prone areas. Thus the subjectCerberus therapeutics may be used to control BMP-4 induced inflammatoryresponse in early steps of atherogenesis in those areas.

During skull development, the cranial connective tissue frameworkundergoes intramembranous ossification to form skull bones (calvaria).As the calvarial bones advance to envelop the brain, fibrous suturesform between the calvarial plates. Expansion of the brain is coupledwith calvarial growth through a series of tissue interactions within thecranial suture complex. Craniosynostosis, or premature cranial suturefusion, results in an abnormal skull shape, blindness and mentalretardation. Recent studies have demonstrated that gain-of-functionmutations in fibroblast growth factor receptors (fgfr) are associatedwith syndromic forms of craniosynostosis. Noggin, an antagonist of bonemorphogenetic proteins (BMPs), is required for embryonic neural tube,somites and skeleton patterning. Warren et al. (Nature. 2003 Apr. 10;422(6932):625-9) show that noggin is expressed postnatally in the suturemesenchyme of patent, but not fusing, cranial sutures, and that nogginexpression is suppressed by FGF2 and syndromic fgfr signalling. Sincenoggin misexpression prevents cranial suture fusion in vitro and invivo, it is suggested that syndromic fgfr-mediated craniosynostoses maybe the result of inappropriate downregulation of noggin expression,leading to abnormally high BMP activity. Thus the subject Cerberustherapeutics may be used to down-regulate BMP activity to prevent ortreat such conditions.

V. Exemplary Formulations

The subject compositions may be used alone, or as part of a conjointtherapy with other compounds/pharmaceutical compositions.

The soluble Coco or Cerberus derivative therapeutics, including theN-terminally truncated Cerberus derivative therapeutics for use in thesubject methods may be conveniently formulated for administration with abiologically acceptable medium, such as water, buffered saline, polyol(for example, glycerol, propylene glycol, liquid polyethylene glycol andthe like) or suitable mixtures thereof. The optimum concentration of theactive ingredient(s) in the chosen medium can be determined empirically,according to procedures well known to medicinal chemists. As usedherein, “biologically acceptable medium” includes any and all solvents,dispersion media, and the like which may be appropriate for the desiredroute of administration of the pharmaceutical preparation. The use ofsuch media for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe activity of the therapeutics, its use in the pharmaceuticalpreparation of the invention is contemplated. Suitable vehicles andtheir formulation inclusive of other proteins are described, forexample, in the book Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences. Mack Publishing Co., Easton, Pa., USA 1985).These vehicles include injectable “deposit formulations.”

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of the Cocoor Cerberus derivative therapeutics suitable for veterinary uses, e.g.,for the treatment of live stock (cow, sheep, goat, pig, and horse, etc.)or domestic animals, e.g., cats and dogs.

Methods of invention may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a therapeutic at a particulartarget site.

The pharmaceutical compositions according to the present invention maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously. The pharmaceutical compositions of thepresent invention may be administered by any means that enables the Cocoor Cerberus derivatives to reach the targeted cells/tissues/organs. Insome embodiments, routes of administration include those selected fromthe group consisting of oral, intravesically, intravenous,intraarterial, intraperitoneal, local administration into the bloodsupply of the organ in which the targeted cells reside or directly intothe cells. Intravenous administration is the preferred mode ofadministration. It may be accomplished with the aid of an infusion pump.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrastermal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,intravesically, nasally, as by, for example, a spray, rectally,intravaginally, parenterally, intracisternally and topically, as bypowders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular therapeutic employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebrovenitricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other non-human mammals such asequines, cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Combined with certain formulations, the subject Coco or Cerberusderivatives can be effective soluble agents. The therapeutic polypeptidecan be provided a fusion peptide along with a second peptide whichpromotes solubility. To illustrate, the Cerberus derivatives of thepresent invention can be provided as part of a fusion polypeptide withall or a fragment of the hinge or Fc portion of the immunoglobulin,which can promote solubility and/or serum stability.

The present invention also contemplates a peptidomimetic sequence of thesubject polypeptide derivatives as described herein.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8^(th) Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”,W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Sources of Caronte and Human Cerberus Protein

Caronte-Fc (Cerberus homolog from Gallus gallus) was ordered from R&DSystems (Minneapolis, Minn.).

Full-length and N-terminally truncated forms of human Cerberus sequencewere cloned into a human CMV derived expression vector, either with orwithout a C-terminal fusion to an Fc portion of IgG1 (both human andmurine IgG1 Fc fusions were produced). These constructs were transientlytransfected in HEK293 cells using polyethylenimine (PEI). Afterculturing, cells were harvested and conditioned media was collected forpurification.

The following constructs were tested:

Human Cerberus, full length, no Fc (SEQ ID NO: 5)

MHLLLFQLLV LLPLGKTTRH QDGRQNQSSL SPVLLPRNQR ELPTGNHEEA EEKPDLFVAV PHLVATSPAG EGQRQREKML SRFGRFWKKP EREMHPSRDS DSEPFPPGTQ SLIQPIDGMK MEKSPLREEA KKFWHHFMFR KTPASQGVIL PIKSHEVHWE TCRTVPFSQT ITHEGCEKVV VQNNLCFGKC GSVHFPGAAQ HSHTSCSHCL PAKFTTMHLP LNCTELSSVI KVVMLVEECQ CKVKTEHEDG HILHAGSQDS FIPGVSA

Human Cerberus, full length, Fc (TGGG linker (SEQ ID NO: 27) and Fc,underlined; native signal sequence underlined with dotted line) (SEQ IDNO: 28)

 QDGRQNQSSL SPVLLPRNQR  ELPTGNHEEA EEKPDLFVAV PHLVATSPAG EGQRQREKML SRFGRFWKKP EREMHPSRDS DSEPFPPGTQ SLIQPIDGMK MEKSPLREEA KKFWHHFMFR KTPASQGVIL PIKSHEVHWE TCRTVPFSQT ITHEGCEKVV VQNNLCFGKC GSVHFPGAAQ HSHTSCSHCL PAKFTTMHLP LNCTELSSVI KVVMLVEECQ CKVKTEHEDG HILHAGSQDS FIPGVSA TGGGTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PVPIEKTISK AKGQPREPQVYTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Human Cerberus, short form, Fc (TGGG linker (SEQ ID NO: 27) and Fc,underlined) (SEQ ID NO: 29)

EVHWETCRTV PFSQTITHEG CEKVVVQNNL CFGKCGSVHF PGAAQHSHTS CSHCLPAKFT TMHLPLNCTE LSSVIKVVML VEECQCKVKT EHEDGHILHA GSQDSFIPGV SA TGGGTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSHEDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTVLHQDWLNGKE YKCKVSNKAL PVPIEKTISK AKGQPREPQVYTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLYS KLTVDKSRWO OGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Three different leader sequences were considered:

(i) Honey bee mellitin (HBML):

(SEQ ID NO: 24) MKFLVNVALVFMVVYISYIYA 

(ii) Tissue Plasminogen Activator (TPA):

(SEQ ID NO: 25) MDAMKRGLCCVLLLCGAVFVSP (iii) Native:

(SEQ ID NO: 26) MLLGQLSTLL CLLSGALPTG S.

A heterologous or native leader sequence may be fused to the proteinsequence at any position within the first 30 amino acids. Modelingsuggests that the native leader would yield a product beginning with“TRH . . . ” at position 18. Analysis of products expressed hereinindicates that the native leader more typically yields a productbeginning “KTT . . . ” at position 16. Therefore, heterologous leadersequences may be fused N-terminal to position 16 or position 18.

Example 2 Caronte Binds GDF-11

GDF-11 is a close homolog of myostatin that regulates neurologicalprocesses. GDF-11 was immobilized on a BiaCore CM5 chip using standardamine coupling procedure. Trace: Caronte (200 μg/ml; R&D Systems) wasinjected on the GDF-11 coupled chip. The tracing in FIG. 4 shows bindingof Caronte to GDF-11.

Example 3 Caronte and Human Cerberus Inhibit GDF-11 andMyostatin-Mediated Signaling

An A-204 Reporter Gene Assay was used to evaluate the effects of Caronteand Cerberus on signaling by GDF-11, myostatin and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100.) SeeFIG. 5. The CAGA12 motif is present in TGF-Beta responsive genes (PAI-1gene), so this vector is of general use for factors signaling throughSmad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 orpGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. In the absence of anyinhibitors, Activin A showed 10 fold stimulation of reporter geneexpression and an ED50˜2 ng/ml. GDF-8: ED50: ˜5 ng/ml, 15 foldstimulation. GDF-11: 16 fold stimulation, ED50: ˜1.5 ng/ml.

As shown in FIG. 6, Caronte inhibits GDF-11 signaling in the A-204Reporter Gene Assay. An ActRIIA-Fc (“IIA muG2a”) fusion also inhibitsGDF-11 signaling. As shown in FIG. 7, Caronte does not inhibit Activin Ain the A-204 Reporter Gene Assay. An ActRIIA-Fc fusion (“IIA muG2a”), asexpected, does inhibit Activin A signaling. Thus, Caronte is a selectiveinhibitor of GDF-11/myostatin while not affecting Activin A signaling.This type of selectivity suggests that Caronte, Cerberus and Coco willhave relatively few side effects when used as a therapeutic. Asexpected, Cerberus behaved much like Caronte, and inhibited myostatinsignaling. See FIG. 8. Similar experiments were conducted to test thebinding of human Cerberus and Coco to Activin A, and these experimentsconfirm that these molecules do not bind to Activin A.

Example 4 Sources of Human Coco Protein

Full-length and N-terminally truncated forms of human Coco were clonedinto a human CMV derived expression vector, either with or without aC-terminal fusion to an Fc portion of IgG1 (both human and murine IgG1Fc fusions were produced). These constructs were transiently transfectedin HEK293 cells using polyethylenimine (PEI). After culturing, cellswere harvested and conditioned media was collected for purification.

The following construct was made, and a murine Fc fusion was made also,and used in the assays presented herein:

Human Coco, full length, Fc (TGGG linker (SEQ ID NO: 27) and mFc) (SEQID NO:22)

MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSW KAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL RNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI EGCHCSPKA TGGGTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSHEDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTVLHQDWLNGKE YKCKVSNKAL PVPIEKTISK AKGQPREPQVYTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Human Coco, short form, Fc (TGGG (SEQ ID NO: 27) linker and mFc) (SEQ IDNO:23)

LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL RNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI EGCHCSPKA TGGGTHTCPP CPAPELLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKAL PVPIEKTISK AKGQPREPQV YTLPPSREEMTKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Three different leader sequences were considered:

(i) Honey bee mellitin (HBML):

(SEQ ID NO: 24) MKFLVNVALVFMVVYISYIYA 

(ii) Tissue Plasminogen Activator (TPA):

(SEQ ID NO: 25) MDAMKRGLCCVLLLCGAVFVSP (iii) Native:

(SEQ ID NO: 26) MLLGQLSTLL CLLSGALPTG S.

Heterologous or native leader sequences may be fused anywhere in thefirst 30 amino acids, and particularly N-terminal to any of amino acids16-23.

Example 5 Human Coco Inhibits GDF-11 Signaling

Conditioned medium from cells expressing human Coco-mFc was tested foreffects on A-204 reporter gene expression in the presence of GDF-11. Theexperiment is analogous to that described in Example 3.

As shown in FIG. 9, conditioned medium containing Coco-mFc inhibitsGDF-11 signaling in the A-204 Reporter Gene Assay, much like Cerberus.Similar experiments showed that Coco-mFc inhibits Nodal signaling.

Example 6 Human Cerberus-Fc is Degraded in Human Serum

The stability of Cerberus polypeptides in the presence of serum wasevaluated. Conditioned medium from cells expressing human full-lengthCerberus-Fc was incubated overnight at 37° C. with varying amounts ofhuman serum (percentages of serum added are shown at top), and resolvedby SDS-PAGE. Western blot (FIG. 10) showed that Cerberus was completelydegraded when incubated with 5% human serum.

N-terminal sequencing of cleavage fragments revealed that proteolysisoccurred at the following sites (cleavage shown by ̂):

(SEQ ID NO: 30)  38 NQR{circumflex over ( )}ELP 43 (SEQ ID NO: 31)138 MFR{circumflex over ( )}KTP 143 (SEQ ID NO: 32) 207 SHC{circumflexover ( )}LPA 212

Example 7 Serum Stable Human Cerberus and Coco Polypeptides

To produce serum-stable Cerberus polypeptides, a variety of mutationsmay be introduced at cleavage sites and the surrounding sequences. Inshort forms of Cerberus, only the L212 cleavage site remains (amino acidnumbering is with reference to the full length, native Cerberussequence, SEQ ID NO: 5), and so a mutation of any, some or all of theamino acids in the sequence SHCLPA (SEQ ID NO: 32) may be altered toeliminate this cleavage site. Generally, mutations will be to small,uncharged groups, such as alanine or serine. Mutations of C211 and/orL212 to serine or alanine are particularly desirable. In addition, or inthe alternative, an N-linked glycosylation site may be introduced at aposition within the range of amino acids 202-222. An N-linkedglycosylation site may also be introduced at a position that is expectedto be proximal to the 212 position in the three-dimensional structure ofthe protein. Similar mutations may be made at each of the other sites 38NQR̂ELP 43 (SEQ ID NO: 30) and 138 MFR̂KTP 143 (SEQ ID NO: 31), dependingon the length of the Cerberus molecule to be employed. A particularlydesirable mutation with respect to the 38 NQR̂ELP 43 (SEQ ID NO: 30)cleavage site is an R to S/T mutation to make the sequence 38 NQ(S/T)ELP43 (SEQ ID NO: 33), simultaneously eliminating the cleavage site andintroducing an N-linked glycosylation site. Additionally, experimentshave shown that products cleaved at E41 and K141 retain myostatinbinding activity. Accordingly, N-terminally truncated forms of Cerberus,beginning at E41 or K141 will be resistant to cleavage at these sitesand retain activity. The activity of the short form suggests that aminimal myostatin binding domain in Cerberus is the cysteine knot,located at amino acids 162-241 of SEQ ID NO:5.

Cerberus constructs with one or more of the alterations (shown inbrackets below; e.g., “[R(T)]” means that an arginine normally at theposition may be replaced with a threonine) will have N-linkedglycosylation sites that will block cleavage and are expected to conferimproved pharmacokinetic properties. The constructs below may beexpressed, for example, with a tPA leader sequence and an Fc sequence.

(SEQ ID NO: 34) TRHQDGRQNQSSLSPVLLPRNQ[R(T)]ELPTGNHEEAEEKPDLFVAVPHLVATSPAGEGQRQREKMLSRFGRFWKKPEREMHPSRDSDSEPFPPGTQSLIQPIDGMKMEKSPLREEAKKFWHHFMF[R(N)]KTPASQGVILPIKSHEVHWETCRTVPFSQTITHEGCEKVVVQNNLCFGKCGSVHFPGAAQHSHTSCSHCLPAKFTTMHLPLNCTELSSVIKVVMLVEECQCKVKTEHEDGHILH[A(N)]GSQDSFIP[G(N)]VSATG 

It is expected that Coco will behave in a manner similar to Cerberus,however, the two likely cleavage sites in Coco occur within the cysteineknot domain at the sequences: 150 PAR̂KRW 155 (SEQ ID NO: 35) and 168SRR̂RVK 173 (SEQ ID NO: 36). Amino acids in these positions may bealtered to eliminate the cleavage, with alanine and serine beingpreferred amino acids. In addition, or in the alternative, an N-linkedglycosylation site may be introduced at or near either of thesepositions. The activity of the short form of Cerberus suggests that aminimal myostatin binding domain of Coco is the cysteine knot, locatedat amino acids 101-185 of SEQ ID NO: 3.

Coco constructs with one or more of the alterations (shown in bracketsbelow) will have N-linked glycosylation sites that will block cleavageand are expected to confer improved pharmacokinetic properties. Theconstructs below may be expressed, for example, with a tPA leadersequence and an Fc sequence.

(SEQ ID NO: 37) GRPEPQSPRPQSWAAANQTWALGPGALPPLVPASALGSWKAFLGLQKARQLGMG[R(N)]L[Q(T)]RGQDEVAAVTLPLNPQEVIQGMCKAVPFVQVFSRPGCSAIRLRNHLCFGHCSSLYIPGSDPTPLVLCNSCMPA[R(N)]K[R(T)]WAPVVLWCLTGSSASR[R(N)][R(A)][V(S)]KISTMLIEGC HCSPKA 

Example 8 Cysteine Variants of Cerberus and Coco

In some proteins, odd numbers of cysteine residues result in a freesulfhydryl group that may cause protein aggregation or otherwiseinterfere with protein production. Both native Coco and native Cerberushave odd numbers of cystein residues. In order to improve the expressionof Cerberus, variants with fewer cysteine residues were generated, aswell as variants with changes in proximity to one or more of thecysteines. Relative to SEQ ID NO: 5, the following variants weregenerated:

C176G; C206G; C223G; N222D.

Each of these proteins were expressible and retained binding to GDF11,indicating that the biochemical activity of these proteins remainedintact. Similar specific variants may be made with respect to Coco(relative to SEQ ID NO: 3):

C115G; C145G; C162G.

Therefore, the disclosure provides Cerberus and Coco variants in whichone or more cysteine residues are deleted or replaced. If replaced, thereplacement amino acid may be any of the other 19 canonical amino acids,although G, A, S and T are preferred.

EQUIVALENTS

A skilled artisan will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated myostatin antagonist protein, the myostatin antagonistprotein comprising the myostatin binding domain of a Coco polypeptide orvariant thereof, which myostatin antagonist protein binds to andneutralizes one or both of nodal and myostatin.
 2. The isolatedmyostatin antagonist protein of claim 1, wherein the myostatinantagonist protein has diminished potency for binding BMP-4 relative toa corresponding wild-type Coco polypeptide.
 3. The isolated myostatinantagonist protein of claim 1, wherein the myostatin antagonist proteincomprises an N-terminally truncated derivative of wild-type Cocoprotein.
 4. The isolated myostatin antagonist protein of claim 1,wherein the myostatin binding domain of Coco comprises the amino acidsequence of SEQ ID NO:
 15. 5. The isolated myostatin antagonist proteinof claim 1, wherein the myostatin antagonist protein binds myostatinwith a K_(D) of 1 μM of less.
 6. The isolated myostatin antagonistprotein of claim 1, further comprising, in addition to the myostatinbinding domain, a heterologous portion.
 7. The isolated myostatinantagonist protein of claim 6, wherein the heterologous portion is animmunoglobulin Fc domain.
 8. The isolated myostatin antagonist proteinof claim 6, wherein the heterologous portion is a polypeptide thatenhances one or more of in vivo stability, in vivo half life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, and/or purification.
 9. The isolated myostatinantagonist protein of claim 8, wherein the heterologous portion is apolypeptide portion is a purification subsequence.
 10. The isolatedmyostatin antagonist protein of claim 9, wherein the purificationsubsequence is an epitope tag, a FLAG tag, a polyhistidine sequence, oras a GST fusion.
 11. The isolated myostatin antagonist protein of claim1, wherein the myostatin antagonist protein comprises one or moremodified amino acid residues, such as a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, or an amino acid conjugated to an organic derivatizing agent.12. The isolated myostatin antagonist protein of claim 1, wherein themyostatin antagonist protein does not substantially inhibit Activin Asignaling in an A204 Reporter Gene Assay.
 13. A method for inhibitingmyostatin signal transduction in an animal, comprising: administering toan animal in need thereof a pharmaceutical preparation comprising aneffective amount of an antagonist myostatin protein comprising themyostatin binding domain of Coco, wherein said pharmaceuticalpreparation is substantially free of pyrogenic materials so as to besuitable for injection as a human or veterinary therapeutic.
 14. Themethod of claim 13, wherein the animal has a pathologic condition, whichis characterized, at least in part, by an abnormal amount, developmentor metabolic activity of muscle or adipose tissue in an animal.
 15. Themethod of claim 13, wherein the animal has a wasting disorder.
 16. Themethod of claim 15, wherein the wasting disorder is selected fromcachexia, anorexia, Duchenne muscular dystrophy (DMD) syndrome, Becker'smuscular dystrophy (BMD) syndrome, acquired immune deficiency syndrome(AIDS) wasting syndrome, age-related wasting, muscular dystrophies, andneuromuscular diseases.
 17. The method of claim 13, wherein the animalhas a metabolic disorder.
 18. The method of claim 17, wherein themetabolic disorder is obesity or type II diabetes.
 19. The method ofclaim 13, wherein the myostatin antagonist protein comprises anN-terminally truncated derivative of wild-type Coco protein.
 20. Themethod of claim 13, wherein the myostatin binding domain of Cococomprises the amino acid sequence of SEQ ID NO:
 15. 21. The method ofclaim 13, wherein the myostatin antagonist protein is a fusion proteincomprising, in addition to the myostatin binding domain of Coco, aheterologous portion.
 22. The method of claim 21, wherein theheterologous portion is a polypeptide that enhances one or more of invivo stability, in vivo half life, uptake/administration, tissuelocalization or distribution, formation of protein complexes, and/orpurification.
 23. The method of claim 21, wherein the heterologousportion is an immunoglobulin Fc domain.
 24. The method of claim 21,wherein the fusion protein has diminished potency for binding BMP-4relative to a corresponding wild-type Coco polypeptide.
 25. The methodof claim 21, wherein the fusion protein binds to and neutralizes one orboth of nodal and myostatin.
 26. The method of claim 21, wherein thefusion protein binds myostatin with a K_(D) of 1 μM of less.
 27. Themethod of claim 23, wherein the fusion protein comprises an additionalpolypeptide portion that enhances one or more of in vivo stability, invivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. 28.The method of claim 27, wherein the additional polypeptide portion is apurification subsequence.
 29. The method of claim 28, wherein thepurification subsequence is an epitope tag, a FLAG tag, a polyhistidinesequence, or as a GST fusion.
 30. The method of claim 21, wherein thefusion protein comprises one or more modified amino acid residues, suchas a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, or an amino acid conjugated toan organic derivatizing agent.
 31. The method of claim 21, wherein thefusion protein does not substantially inhibit Activin A signaling in anA204 Reporter Gene Assay.